The subject invention pertains to the field of genetics, more particularly to the genetic manipulation of cells.
When conducting genetic manipulations or other treatments of cells, pollen, organs, or tissue, it is often necessary to partially or completely penetrate the cell walls and/or membranes with a penetrating member to facilitate delivery of biological or other agents. This penetration is necessary to achieve a desired effect on the cell wall and/or internal cellular elements such as the cytoplasm, nucleus, plastids, chromosomes, plasmids, etc. The objective of such a procedure may be, for example, the destruction of selected elements or the production of new or improved biological characteristics. These procedures can be used, for example, to improve by modifying plants, animals, or microbes growth rate, disease resistance, or protein and secondary metabolite production. Other applications include the tagging of cells for tracking and identification, or subsequent micromanipulation over space and time.
Often penetration of cells is accomplished by applying the biological or other agent to carrier particles that are introduced into the cells. Such methods are well known in the art.
In genetic research, for example, such methods are used to penetrate tissue and cells with particles precoated with DNA encoding genes of interest; cell penetration is followed by DNA delivery into the cell nucleus. To reach the intracellular space and then the cell nucleus, the particles must traverse formidable cell walls and membranes. Because these cell walls are difficult to penetrate, the particles carrying the DNA are driven into the cells by the force of an explosive or an electrical discharge. This causes the kinetically driven particles to smash into the target tissue. Even then, to have the necessary energy for penetration, the particles must be several micrometers in diameter. Thus, the implantation process results in appreciable cell damage due to the impact of the particles and/or sonic concussion from the particle-propelling discharge. Some cell tissue, drawing upon its natural strength, may sufficiently recover from this trauma and integrate the newly delivered genetic material into its chromosomes. Other tissue, however, is unable to do so.
These prior methods of delivering particles to cells also lack sufficient control over particle size distribution, particle coating quality, and the velocity and direction of travel of the particles. This results in a lack of predictability and reproducibility of the particle delivery technique. Prior delivery techniques are further disadvantaged because they require the target tissue be maintained in a vacuum, required to penetrate several cell layers deep, which removes moisture from the treated tissue contributing to tissue degradation. In addition, to set up the apparatus for performing the implantations requires time-consuming labor prior to each implantation cycle. Clean up is also time consuming and laborious. As such, the throughputs of the apparatus are relatively low.
Other methods employed or suggested for direct gene delivery to cells include the use of microlasers, microbead vortexing, electrofusion, chemical fusion, microinjection, xe2x80x9cwhiskers,xe2x80x9d and electroporation. Such techniques all rely on increasing the permeability of the tissue cells by physically, chemically, or electrically disrupting cell walls and/or membranes temporarily so that exogenously added DNA may then enter the cell through the temporary ruptures. Some of these methods, including microinjection and fusion of preselected protoplasts or subprotoplasts, require working at the single cell level. This necessitates micromanipulation of the cells, often involving immobilization by agarose plating or pipette suction. Such micromanipulations must be carried out with a microscope placed in the sterile environment of a laminar flow hood, which can be very cumbersome. As another example, controlled fusion in the production of somatic hybrids, requires bringing the fusion partners into close proximity which is technically difficult to accomplish. Other techniques, such as use of fragile silica-based fibers or xe2x80x9cwhiskers,xe2x80x9d involves rapid gyratory or oscillatory motion which obviates directional delivery and re-use of the now fragmented fibers. Such techniques are restricted to use with one or a few types of cells, subcellular targets, or tissues and rarely, if at all, across taxonomic kingdoms, and exclude treatment of tissues in situ.
U.S. Pat. No. 5,516,670 discloses a magnetophoretic method and apparatus for delivering small, needle-shaped, magnetizable particles to a target site. The particles serve as carriers for chemical substances such as DNA, antibodies, or pharmaceuticals which are then presented to the target material in the hope of causing an interaction with said material and bringing a beneficial change to the target host.
The decisive feature described in the aforementioned patent is the ability to move magnetizable particles forward towards the point of convergence of the conical magnetic field, whose gradient along the length of the particle provides the force differential that propels the particle while orienting it vector-like towards the target. In other words, the length of the particle, its magnetizable mass, its magnetic saturation potential, and the magnetic flux density difference between the tip of the acicular particle and its tail where the flux density is less, represent the conditions that collectively result in the force that drives the magnetizable particles into the target site. To obtain the necessary force to overcome the mechanical target resistance such as represented by a cell wall or skin, one endeavors to use powerful magnetic field gradients, long needle-shaped micromagnets of high coercive strength, and sharp tips of the particle to obtain the highest possible penetration pressure at the point where the tips push against said cell walls. These aforementioned conditions determine the mechanical and electrical engineering design of the apparatus, including its electrical energy requirement and, ultimately, its weight and cost.
The main object of the present invention is to reduce the weight and power consumption of such a particle injection apparatus and turn it into a device that is portable to the extent that it can fit into one""s pocket and operate without electrical power. To achieve this goal, various attributes and features must be incorporated in a novel device that differs in its very fundamental aspects from what is disclosed in U.S. Pat. No. 5,516,670.
All patents and other publications cited herein are incorporated by reference in their entirety, to the extent not inconsistent with the explicit teachings set forth herein.
The subject invention pertains to a method of magneto-mechanical insertion and retraction of magnetizable particles into a target material by means of a magneto-mechanical delivery device. The device delivers substances such as DNA, chemicals and the like. The subject method and device is utilized for the delivery of substances by effecting the penetration of particles into a target body. Such penetration of a target initiates the interaction between the material contained within the target site and the substance delivered by the penetrating particle. In a preferred embodiment, the subject device is portable and does not require electrical power. The subject magneto-mechanical apparatus can be loaded with reactive substances which are delivered to the target material. Such reactive substances can be carried by needle-like, magnetizable particles, wherein the particles are loaded into one side of the subject device and stand erect and parallel on a loading surface ready to penetrate a target site. The substantially parallel, erect position of the particles is achieved by positioning a magnet such that the field lines of the magnet extend perpendicularly from the substrate, causing the substantially parallel, erect orientation of the particles. The loading surface is moved by mechanical or other means towards a second substrate on which the target material is placed. The two substrates are mated together by a strong non-magnetic force.
Accordingly, it is an object of the present invention to provide a magneto-mechanical delivery service and method for using same.
It is a further object of the present invention to provide a magneto-mechanical delivery device and method for using same that is portable.
It is a still further object of the present invention to provide a portable magneto-mechanical delivery device and method for the delivery of substances to target materials.
It is a still further object of the present invention to provide a portable magneto-mechanical delivery device and method for the genetic manipulation of cells.
Further objects and advantages of the present invention will become apparent by reference to the following detailed disclosure of the invention and appended drawings.